The goal of this project is to develop new therapeutic strategies to improve myocardial tolerance to ischemia by targeting the heart's endogenous defense mechanisms against oxidative stress. The specific aim of the proposed research is to determine the mechanism(s) whereby adenosine and adenosine receptor activation reduce cellular and subcellular reactive oxygen species (RO2) formation and oxidative stress; and the role adenosine plays in enhancing endogenous antioxidant defense mechanisms. Studies will be performed in isolated rat ventricular myocytes and a porcine in vivo regional ischemia preparation. In the in vivo porcine studies, the cardiac microdialysis technique will be used to measure interstitial fluid levels of ROS. Contractile dysfunction will be evaluated by load insensitive measures of regional contractility. In myocytes, RO2 will be measures with the fluorescent probe dichlorofluorescein diacetate, contractility. In myocytes, ROS will be measured with the fluorescent probe dichlorofluorescein diacetate, and mitochondrial oxidative stress will be estimated by monitoring mitochondrial flavoprotein autofluorescence. Oxidative stress and cell function will be measured in myocytes submitted to stimulated ischemia and reperfusion. Antioxidant levels will be determined by measuring reduced glutathione, oxidized glutathione and the NADPH/NADP+ ratio. The effects of adenosine receptor agonists, alone and in combination of oxidative stress and antioxidant status will be correlated with myocyte twitch amplitude and the extent of stunning and cell function following hypoxia-reoxygenation. The findings could lead to the development of new therapeutic strategies to improve hemodynamic performance and survival in patients with ischemic heart disease.